Retroviral vector particles are useful agents for introducing polynucleotides into cells, such as eukaryotic cells. The term "introducing" as used herein encompasses a variety of methods of transferring polynucleotides into a cell, such methods including transformation, transduction, transfection, and transinfection.
Retroviruses typically have three common open reading frames, gag, pol, and env, which encode the structural proteins, encode enzymes including reverse transcriptase, and encode an envelope protein, respectively. Typically, retroviral vector particles are produced by packaging cell lines that provide the necessary gag, pol, and env gene products in trans. (Miller, et al., Human Gene Therapy, Vol. 1, pgs. 5-14 (1990)). This approach results in the production of retroviral vector particles which transduce mammalian cells, but are incapable of further replication after they have integrated into the genome of the cell.
Thus, retroviral vector particles have been used for introducing polynucleotides into cells for gene therapy purposes. In one approach, cells are obtained from a patient, and retroviral vector particles are used to introduce a desired polynucleotide into the cells, and such modified cells are returned to the patient for a therapeutic purpose. See Anderson et al., U.S. Pat. No. 5,399,346, issued Mar. 21, 1995. In another approach, retroviral vector particles may be administered to the patient in vivo, whereby the retroviral vector particles transduce cells of the patient in vivo.
In many gene therapy protocols, it would be desirable to target retroviral vector particle infection to a specific population of cells either in vivo or in vitro. In such circumstances, the broad host range of typical retroviruses presents a significant problem. A key determinant of viral host range is the "envelope" or "env" protein (encoded by the env gene) which is involved in binding to receptors on the surface of susceptible cells. Where it is possible to purify the desired target cells, either before or after transduction, such purification necessitates undesirable manipulations of the cells and may be problematic in situations in which the preferred target cells either are difficult to purify or are present at low or variable frequencies in mixed cell populations. Thus, it would be advantageous to have retroviral vector particles which could infect particular types of mammalian cells.
Generation of a targeting retroviral vector particle would enable the recombinant retrovirus to deliver a therapeutic gene to the target tissue through cross-species and/or tissue-specific infection. To achieve such a goal, common strategies have been to modify the natural host range determinant, the retroviral envelope protein, by inserting new receptor binding polypeptides into the surface domain (SU) of the envelope protein. The envelope protein, however, is difficult to modify. Prior attempts to modify the retroviral envelope have been directed to the insertion of targeting polypeptides into the receptor binding region of the envelope protein. Such attempts have resulted in the disruption of the envelope structure to such an extent that the folding, processing, and incorporation of the envelope protein is impaired strongly. Also, the activity of the inserted polypeptide may be limited by the steric hindrance caused by the surrounding host protein residues.